The invention relates generally to clonal primate mesenchymal progenitors and to mesenchymal stem cell (MSC) lines and methods for identifying and generating such cells, and more particularly to methods for generating clonal mesenchymal progenitors and MSC lines under serum-free conditions.
MSCs can differentiate into at least three downstream mesenchymal cell lineages (i.e., osteoblasts, chondroblasts and adipocytes). To date, no unique MSC marker has been identified. As such, morphological and functional criteria are used to identify these cells. See, Horwitz E, et al., “Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement,” Cytotherapy 7:393 (2005); and Dominici M, et al., “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement,” Cytotherapy 8:315 (2006). Because MSCs can differentiate into many cell types, the art contemplates methods for differentiating MSCs for cell-based therapies, for regenerative medicine and for reconstructive medicine.
Typically, MSCs are isolated from adult bone marrow, fat, cartilage and muscle. Pittenger F, et al., “Multilineage potential of adult human mesenchymal stem cells,” Science 284:143-147 (1999); Zuk P, et al., “Multilineage cells from human adipose tissue: implications for cell-based therapies,” Tissue Eng. 7:211-228 (2001); and Young H, et al., “Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors,” Anat. Rec. 264:51-62 (2001). MSCs have also been isolated from human peripheral blood. Kassis I, et al., “Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads,” Bone Marrow Transplant. 37:967-976 (2006). MSCs can also be isolated from human neonatal tissue, such as Wharton's jelly (Wang H, et al., “Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord,” Stem Cells 22:1330-1337 (2004)), human placenta (Fukuchi Y, et al., “Human placenta-derived cells have mesenchymal stem/progenitor cell potential,” Stem Cells 22:649-658 (2004)); and umbilical cord blood (Erices A, et al., “Mesenchymal progenitor cells in human umbilical cord blood,” Br. J. Haematol. 109:235-242 (2000)) and human fetal tissues (Campagnoli C, et al., “Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow,” Blood 98:2396-2402 (2001)).
The art is limited by an inability to isolate sufficient MSCs for subsequent differentiation and use. Where suitable donors are available, the invasive procedures required to isolate even a limited number of cells present risks to donors. It also remains difficult to maintain isolated MSCs in long-term culture and to maintain such cultures free of bacterial or viral contamination.
Efforts to devise methods for differentiating embryonic stem cells (ESCs) including human ESCs (hESCs) to MSCs either have required culturing the cells in a medium containing potentially contaminating serum or have yielded cells that retain characteristics of undifferentiated hESCs. For example, Barberi et al. differentiated hESCs to MSCs on mitotically-inactivated mouse stromal cell lines (i.e., feeder cells) with 20% heat-inactivated fetal bovine serum (FBS) in alpha MEM medium for 40 days. Barberi T, et al. “Derivation of multipotent mesenchymal precursors from human embryonic stem cells,” PLoS Med. 2:e161 (2005). Cells were harvested and assayed for CD73, and CD73+ cells were then plated in the absence of the feeder cells with 20% FBS in alpha MEM for 7 to 10 days. Barberi et al. differentiated the MSCs into adipogenic cells, chondrogenic cells, osteogenic cells and myogenic cells.
Likewise, Olivier et al. differentiated hESCs to MSCs by plating raclures (i.e., spontaneously differentiated cells that appear in hESC culture in the center or at the edges of colonies) with D10 medium (DMEM, 10% FBS, 1% penicillin/streptomycin and 1% non-essential amino acids) changed weekly until a thick, multi-layer epithelium developed. Olivier E, et al., “Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells,” Stem Cells 24 1914-1922 (2006). After approximately four weeks, MSCs were isolated by dissociating the epithelium with a mixture of trypsin, collagenase type IV and dispase for four to six hours, followed by re-plating in D10 medium. Olivier et al.'s MSCs grew robustly, had stable karyotypes, were contact inhibited, senesced after twenty passages and differentiated into adipogenic and osteogenic cells. Olivier et al. did not report that the cells differentiated into chondroblasts. Unlike Barberi et al., Olivier et al. did not require feeder cells to support differentiation of hESC to MSCs. However, Olivier et al.'s MSCs were SSEA-4 positive, suggesting that these MSCs expressed cell surface markers characteristic of hESC.
Pike & Shevde differentiated hESCs to MSCs via embryoid bodies (EBs) incubated for ten to twelve days in a mesenchymal-specific medium (MesenCult® medium with 10% FBS; alpha MEM with glutamine and nucleosides; or DMEM with glucose and glutamine, replaced every two days). US Patent Publication No. 2006/0008902. The EBs were digested, and pre-mesenchymal cells were cultured to 80% confluence. The cells were trypsinized and passaged three times in mesenchymal-specific medium.
Meuleman et al. reported culturing MSCs in a serum-free medium; however, it was later discovered that the medium did in fact contain animal serum as a component. Meuleman N, et al., “Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-minimal essential medium (MEM),” Eur. J. Haematol. 76:309-316 (2006); and Meuleman N, et al., “Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-minimal essential medium (MEM),” Eur. J. Haematol. 77:168 (2007); but see, Korhonen M, “Culture of human mesenchymal stem cells in serum-free conditions: no breakthroughs yet,” Eur. J. Haematol. 77:167 (2007).
Those methods cultured and differentiated MSCs in serum-containing medium. Serum-free conditions for culturing and differentiating MSCs, if defined, would reduce variation among batches and eliminate a risk of infection transmitted by xenogenic by-products and pathogens. Sotiropoulou P, et al., “Cell culture medium composition and translational adult bone marrow-derived stem cell research,” Stem Cells 24:1409-1410 (2006).
For the foregoing reasons, there is a need for new methods for obtaining early mesenchymal progenitors and MSCs, especially when derived under serum-free conditions.